Methods for identifying inhibitors of methionine aminopeptidases

ABSTRACT

Methods are provided for detecting methionine aminopeptidase (MAP) activity and for detecting inhibitors of MAP. The methods utilize a peptide comprising an N-terminal methionine which can be cleaved from the peptide by MAP, and a C-terminal detection moiety which is released by a second peptidase only if the N-terminal methionine has been cleaved from the peptide. When the peptide is combined with MAP and the second peptidase, the detection moiety is released, while the addition of a MAP inhibitor will inhibit the release of the detection moiety. Reaction mixes, peptides, and kits which are useful for practicing the methods are also provided.

This invention was made with Government support under National ScienceFoundation Grant No. MCB 9512655. The Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention generally relates to assays for enzyme activityand for enzyme inhibitors. More specifically, the invention relates toassays for detecting methionine aminopeptidases and inhibitors ofmethionine aminopeptidases.

(2) Description of the Related Art

In all living cells, protein synthesis is initiated with an AUG codon,specifying methionine as the N-terminal amino acid in nascent proteins.In both prokaryotes and eukaryotes, this N-terminal methionine will beremoved by a methionine aminopeptidase (MAP) (EC 3.4.11.18) if thepenultimate amino acid residue is small and uncharged, e.g., Gly, Ala,Ser, Cys, Thr, Pro, and Val, although methionine cleavage activity byMAP is reduced when the N-terminal three amino acids are Met-Thr-Pro orMet-Val-Pro (Moerschell et al., 1990, J. Biol. Chem. 265, 19638-19643;Tsunasawa et al., 1985, J. Biol. Chem. 260, 5382-5391). Removal of theN-terminal methionine is essential for certain proteins to functionnormally in vivo. For example, the removal of the initiator methionineis often required for subsequent N-terminal modifications, such asN-myristoylation, which is essential for the normal function of varioussignal transduction proteins, cancer cells, protein targeting moieties,and enzymes (Gordon et al., 1991, J. Biol. Chem. 266, 8647-8650; Duronioet al., 1989, Science 243, 796-800).

Methionine aminopeptidases have been isolated and cloned from severalorganisms, including E. coli and several other eubacteria, yeast, rat,and various archaea. Currently discovered MAPs have been categorizedinto two types, type 1 MAP and type 2 MAP, based on structural andsequence similarities. Eubacteria have type 1, archaea have type 2, andeukaryotes have both types. In eukaryotes, null mutants in either typeare viable but slow growing, but null mutants of both MAP types arenonviable (Li and Chang, 1995, Proc. Natl. Acad. Sci. USA 92,12357-12361; Li and Chang, 1996, Biochem. Biophys. Res. Commun. 227,152-159; Bradshaw et al., 1998, Trends Biochem. Sci. 21, 285-286).Similarly, knockouts of the bacterial MAP1 gene are lethal (Ben-Bassatet al., 1987, H. Bacteriol. 169, 751-757). Thus, MAP activity isessential for normal functioning of prokaryotic and eukaryotic cells.

Aside from their role in cleaving the initiator methionine of proteins,MAPs affect other cellular functions. For example, human type 2 MAP alsoserves as eukaryotic initiation factor-2, which regulates proteinsynthesis (U.S. Pat. No. 5,885,820). Also, the mode of action offumagillin-type angiogenesis inhibitors is the irreversible inhibitionof type 2 MAP (Griffith et al., 1997, Chem. Biol. 4, 461-471; Liu etal., 1998, Science 282, 1324-1327; Lowther et al., Proc. Natl. Acad.Sci. USA 95, 12153-12157; Sin et al., 1997, Proc. Natl. Acad. Sci. USA94, 6099-6103) thus indicating an essential role of type 2 MAP inangiogenesis.

Because of the crucial role of MAPs in prokaryotic and eukaryoticfunctions, there is an interest in the discovery of additionalinhibitors of these enzymes, which may serve as antibiotics or aschemotheraputic agents which inhibit angiogenesis in tumors. However,current methods for monitoring MAP activity are inadequate for thistask. These methods include the colorimetric ninhydrin method (Doi etal., 1981, Anal. Biochem. 118, 173-184); amino acid oxidase treatmentfollowed by peroxidase reaction and o-dianisidine color development(Carter and Miller, 1984, J. Bacteriol. 159, 453-459); amino acidanalysis via ion exchange chromatography followed by postcolumnderivatization with ninhydrin (Moore et al., 1958, Anal. Biochem. 30,1185-1190), fluorescamine (Stein et al., 1973, Arch. Biochem. Biophys.155, 202-212), or o-phthalaldehyde/β-mermercaptoethanol (Roth, 1971,Anal. Chem 43, 880-882); precolumn derivatization of amino acidsfollowed by reverse phase HPLC chromatography (Cohen and Strydom, 1988,Anal. Biochem. 174, 1-16; Zuo et al., 1994, Anal. Biochem. 222,514-516); and separation of substrate peptides and products by reversephase HPLC with on-line UV detection of each separated compound(Larrabee et al., 1999, Anal. Biochem. 269, 194-198; Walker et al.,1999, Biotechnol. Appl. Biochem. 29, 157-163). These methods are eithernot sensitive or accurate enough for quantitative assays, or are notrapid enough for high-throughput screening procedures. Thus, there is aneed for new assays for MAP activity which are rapid and quantitativeenough for use in procedures requiring high-throughput MAP analysis,such as screening for MAP inhibitors.

SUMMARY OF THE INVENTION

Accordingly, the inventor has succeeded in inventing a novel MAP assaywhich is rapid, quantitative, and suitable for automated procedures. Theassay employs a second peptidase and a peptide which comprises anN-terminal methionine which can be cleaved by MAP, along with aC-terminal detection moiety which can be released from the peptide bythe second peptidase only if the N-terminal methionine has been cleavedfrom the peptide.

Thus, one embodiment of the invention is directed to methods fordetecting methionine aminopeptidase (MAP) activity in a composition.These methods comprise (a) combining the composition with a peptidecomprising an N-terminal methionine under conditions that the N-terminalmethionine can be cleaved from the peptide by a MAP to produce a cleavedpeptide, wherein the peptide contains a C-terminal detection moietywhich is released by a second peptidase only if the N-terminalmethionine has been cleaved from the peptide; (b) reacting any cleavedpeptide produced in (a) with the second peptidase to release thedetection moiety; and (c) detecting any detection moiety released. Apreferred second peptidase is dipeptidyl peptidase IV. When dipeptidylpeptidase IV is utilized, a preferred peptide comprises Met-X_(aa)-Pro,wherein X_(aa) is Ala, Cys, Gly, or Ser; a most preferred peptide isMet-Gly-Pro-p-nitroanilide.

The present invention is also directed to methods for determiningwhether a substance inhibits a MAP. The methods comprise (a) combiningthe substance, the MAP, and a peptide comprising an N-terminalmethionine under conditions that the N-terminal methionine can becleaved from the peptide by the MAP to produce a cleaved peptide,wherein the peptide contains a C-terminal detection moiety which isreleased by a second peptidase only if the N-terminal methionine hasbeen cleaved from the peptide; (b) reacting any cleaved peptide producedin (a) with the second peptidase to release the detection moiety; and(c) detecting any detection moiety released. As with the previouslydescribed method, a preferred second peptidase is dipeptidyl peptidaseIV, and a preferred peptide is Met-Gly-Pro-p-nitroanilide. This methodalso preferably comprises quantitating the amount of detection moietyreleased, in order to more accurately detect MAP inhibitors.

In another embodiment, the present invention is directed to reactionmixtures suitable for use in the methods described above. The reactionmixture comprises (a) a peptide comprising an N-terminal methioninewhich can be cleaved by the methionine aminopeptidase, and a C-terminaldetection moiety which can be released by a second peptidase only if theN-terminal methionine has been cleaved from the peptide; and (b) thesecond peptidase. A MAP may also be included in the mixture.

In an additional embodiment, the present invention is directed to a kitfor performing the methods described above, comprising the secondpeptidase and the peptide described in those methods, along withinstructions for performing the methods. Preferably, the kit alsocomprises a MAP.

The present invention is also directed to the peptides described in theabove embodiments.

Among the several advantages achieved by the present invention,therefore, may be noted the provision of methods and reagents forrapidly detecting and quantifying MAP activity and MAP inhibitoractivity. These methods are more suitable than previously known methodsfor rapid screening and quantifying MAP and MAP inhibitor activitybecause they combine speed, quantitative accuracy, and potential forautomated execution, which has not been previously achieved.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts reactions catalyzed by MAP (A) and dipeptidyl peptidaseIV (B) which are useful in the present invention.

FIG. 2 is a graph depicting the results of an assay for MAP according tothe invention, using the peptide Met-Gly-Pro-p-nitroanilide, wheredipeptidyl peptidase IV is able to release p-nitroanilide in thepresence of type 1 MAP (•—•) and type 2 MAP (∘—∘), but not withdipeptidyl peptidase IV alone ({overscore (V)}—{overscore (V)}).

FIG. 3 is a graph depicting the results of an assay for inhibition ofMAP by fumagillin according to the present invention, using dipeptidylpeptidase IV and the peptide Met-Gly-Pro-p-nitroanilide, wherefumagillin strongly inhibited release of p-nitroanilide when type 2 MAPwas used ({overscore (V)}—{overscore (V)}), but not when type 1 MAP wasused (∘—∘), or when dipeptidyl peptidase IV was used withGly-Pro-p-nitroanilide (•—•).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to rapid and quantitative MAP assays.These assays employ a second peptidase and a peptide, wherein thepeptide comprises an N-terminal Met which is capable of being cleaved byMAP, and a C-terminal detection moiety which is capable of beingreleased from the peptide by the second peptidase only if the N-terminalMet has been cleaved from the peptide. The detection moiety is notdetectable when it is covalently bound to the peptide but becomesdetectable upon its release from the peptide.

It has been known that peptide analogs such as Leu-p-nitroanilide serveas a substrate for aminopeptidases such as leucine aminopeptidase (EC3.4.11.1). When conjugated to Leu, p-nitroanilide absorbs much lesslight at 405 nm than when released from the amino acid. Thus, cleavageof Leu-p-nitroanilide by leucine aminopeptidase to form leucine+p-nitroanilide is accompanied by a quantitative increase in A₄₀₅.However, MAP cannot cleave Met-p-nitroanilide to release p-nitroanilide.

It is also known that other enzymes are able to use a short peptideconjugated to p-nitroanilide or other such detection moieties as asubstrate, releasing p-nitroanilide. One notable example is dipeptidylpeptidase IV (EC 3.4.14.5)(also known as leukocyte differentiationantigen CD26) which will cleave X_(aa)-Pr-p-nitroanilide, where X_(aa)is any amino acid, to release p-nitroanilide (e.g., reaction B in FIG.1). Dipeptidyl peptidase IV can also utilize Ala and hydroxyproline asthe penultimate amino acid (in place of Pro), albeit with slower releaseof the detection moiety (Ikehara et al., 1994, Meth. Enzymol. 244,215-227). It has also recently been discovered that dipeptidyl peptidaseIV can utilize an N-terminal Tyr-Gly as a substrate (Proost et al.,1999, J. Biol. Chem. 274, 3988-3993).

It has now been discovered that, when either type 1 or type 2 MAP ismixed with Met-Gly-Pro-p-nitroanilide and dipeptidyl peptidase IV,p-nitroanilide is released from the peptide, whereas no p-nitroanilideis released when MAP is not included (FIG. 2). Thus, MAP is able toutilize a tripeptide as a substrate, mediating, e.g., reaction A in FIG.1 to create Gly-Pro-p-nitroanilide, which dipeptidyl peptidase IV canuse as a substrate to release p-nitroanilide.

As used herein, the term “release”, when referring to a detection moietyon a peptide, means that the covalent bond between the detection moietyand the peptide has been broken, generally by enzymatic action.

Thus, in some embodiments of the invention, methods for detecting MAPactivity in a composition is provided. These methods comprise combiningthe composition with a peptide comprising an N-terminal methionine underconditions that the N-terminal methionine can be cleaved, wherein thepeptide also contains a C-terminal detection moiety which can bereleased by a second peptidase only if the N-terminal methionine hasbeen cleaved. Any cleaved peptide produced upon reaction with thecomposition is reacted with the second peptidase to release thedetection moiety, which is then detected.

The second peptidase must be able to release the detection moiety fromthe peptide when the N-terminal methionine of the peptide has beenremoved. Additionally, the second peptidase must not be able to releasethe detection moiety from the peptide when an N-terminal Met is present.Also, since MAP can cleave the N-terminal Met from a peptide only whencertain amino acids (e.g., Gly, Ala, Ser, Cys, Thr, Pro, or Val arepresent in the penultimate position of the peptide), thepeptide-detection moiety which can be cleaved by the second peptidasemust have one of those amino acids in the N-terminal position.Preferably, the second peptidase is dipeptidyl peptidase IV, which canutilize, e.g., Gly-Pr-p-nitroanilide as a substrate to releasep-nitroanilide. Note that the N-terminal amino acid of this dipeptidylpeptidase IV substrate is Gly, which is a permissible penultimate aminoacid for MAP. Thus, Met-Gly-Pro-p-nitroanilide can be cleaved by MAP torelease Gly-Pro-p-nitroanilide, a dipeptidyl peptidase IV substrate, butMet-Gly-Pro-p-nitroanilide itself is not a dipeptidyl peptidase IVsubstrate. Therefore, dipeptidyl peptidase IV is a suitable secondpeptidase for this method. Other peptidases may also be useful as thesecond peptidase, for example triaminopeptidase, which can utilizeGly-Pro-Leu-detection moiety as a substrate (Aoyagi et al., 1978,Biochem. Biophys. Res. Commun. 80, 435), and cathepsin C, which canutilize Gly-Phe-detection moiety as a substrate (Jadot et al., 1984,Biochem. J., 219, 965; Doughty and Gruenstein, 1986, Biochem. and CellBiol., 64, 772).

Preferably, the second peptidase is active under the same conditions asMAP (e.g., 10 mM Hepes, pH 7.35, 1.5 mM MgCl₂, 150 mM KCl, 10% glycerol,0.1-0.5 mM Co²⁺ [Buffer H], [Zuo et al., supra], 30-35° C.), to allowboth peptidase reactions to occur without adjusting conditions. Such asecond peptidase would allow the assay to be performed in one step,where the composition, peptide, and second peptidase are incubatedsimultaneously.

As previously discussed, the peptide to be used in the assay must havean N-terminal methionine which can be cleaved by MAP, i.e., it must havea penultimate amino acid which permits MAP to cleave the N-terminalmethionine (e.g., Gly, Ala, Ser, Cys, Thr, Pro, or Val). The peptidemust also have a C-terminal detection moiety which can be cleaved fromthe peptide by the second peptidase, only if the N-terminal methionineis not present. Thus, the sequence must permit such cleavage. When thesecond peptidase is dipeptidyl peptidase IV, permissible peptidesinclude Met-X_(aa1)-X_(aa2)-detection moiety, where X_(aa1) is Gly, Ala,Ser, Cys, Thr, Pro, or Val, preferably Ala, Cys, Gly, or Ser, mostpreferably Gly; and X_(aa2) is Pro, Hyp, or Ala, most preferably Pro.Thus, the most preferred peptide for dipeptidyl peptidase IV isMet-Gly-Pro-detection moiety. However, any peptide which will result inrelease of the detection moiety when active MAP is present but not whenMAP is absent can be useful in these methods. For example, multiples ofthe peptide cleavable by dipeptidyl peptidase IV may be used, forexample Met-Gly-Pro-Gly-Pro-detection moiety, orMet-Gly-Pro-Gly-Pro-Gly-Pro-detection moiety, since dipeptidyl peptidaseIV is capable of cleaving each successive dipeptide (after N-terminalMet cleavage by MAP) to release the detection moiety. The peptides ofthe present invention can be made by any method known in the art.

These methods can also employ a third enzyme which, in combination withMAP and the second peptidase (but not without MAP) releases or activatesa detection moiety. In this scheme, the peptide is designed toaccommodate the third enzyme. For example, the second peptidase andthird enzyme can be dipeptidyl peptidase IV and cathepsin C, and thepeptide can be Met-Gly-Pro-Gly-Phe-detection moiety orMet-Gly-Phe-Gly-Pro-detection moiety, where the second peptidase and thethird enzyme work successively. Alternatively, the third enzyme can acton the detection moiety to produce a detectable signal. In that case,the detection moiety is a substrate for the third enzyme, for exampleluciferin for a luciferase third enzyme.

The invention is not narrowly limited by choice of detection moiety,provided the detection moiety is capable of being released from thepeptide to produce a detectable signal. The detection moiety can be amoiety which can be determined by a secondary step, for example by achromatographic, centrifugal, or electrophoretic separation, or byenzyme reaction (as previously discussed). In this regard, the detectionmoiety can itself be an enzyme, provided that (1) the enzyme hasincreased activity when released from the peptide by the secondpeptidase, and (2) the enzyme has activity for a substrate which can bedetected after the enzyme acts on it. Preferably, the detection moietyis one that can be easily detected, for example by visual, photometric,spectrometric, or fluorescent means. Non-limiting examples of suchmoieties are cresyl violet, which is fluorescent when released (VanNoorden et al., 1997, Anal. Biochem. 252, 71-77);7-amino-3-trifluoromethylcoumarine, also fluorescent when released(Lojda, 1996, Acta Histochem 98, 215-218); 4-methoxy-2-naphthylamine(Scharpe et al., 1988, Clin. Chem. 34, 2299-2301) or 2-naphthylamine(Ikehara et al., supra), which are also fluorescent upon release;1-hydroxy-4-naphthylamide, which, when released, react with tetrazoliumsalts to form a water-insoluble, deeply colored formazans (useful forcertain solid phase formats of the method);3,5-dibromo-4-hydroxyanilide, which forms2,6-dibromophenol-indo-p-xylenol upon release, a compound with anabsorption maxima at 600 nm (Shibuya-Saruta et al., 1995, J. Clin. Lab.Anal. 9, 113-118); and p-nitroanilide, which has an absorption maxima at415 nm when released (Id.). A preferred detection moiety isp-nitroanilide, because it has been widely used, e.g., as a detectionmoiety in dipeptidyl peptidase assays (using, e.g., the peptideGly-Pro-p-nitroanilide). Several different detection methods have beenused to detect p-nitroanilide (Ikehara, supra). For example, thiscompound can be visualized, or measured directly photometrically orspectrometrically at, e.g., 385 nm. A more sensitive p-nitroanilidedetection method involves diazotization and coupling withN-(1-naphthyl)ethylenediamine to produce a product which can be measuredat 548 nm (Id).

The invention encompasses various assay formats. For example, the assaycan be performed in a two-step method, by first mixing the composition(where MAP may be present) with the peptide, allowing any MAP present tocleave the N-terminal methionine from the peptide, then adding thesecond peptidase, allowing the detection moiety to be released. Thisformat may be useful where the second peptidase has differentrequirements for activity (e.g., different pH or temperature optima),where the conditions can be changed prior to adding the secondpeptidase. Preferably, however, the assay is performed in one step,where the composition, the peptide, and the second peptidase are mixedtogether and incubated to allow the detection moiety to be released.

The methods can also be performed wholly in an aqueous solution (e.g.,in tubes or microtiter wells), or partly or wholly on a solid phase. Thesolid phase can be employed, e.g., where a peptidase is adsorbed orcovalently bound to a bead, tube, or well and the other components arein solution, or where the entire assay is performed on a solid phasesuch as a nitrocellulose membrane, and the detection moiety produces aninsoluble detectable product upon release from the peptide. Such a solidphase format can also be used in histochemical applications to localizeMAP present in tissue. In those applications, the peptide along with thedipeptidyl peptidase IV is applied to the tissue. Preferably, thedetection moiety will be insoluble when released from the peptide, toprevent the MAP “signal” from moving from the MAP location in thetissue.

These methods can be used qualitatively or quantitatively. For example,the methods can be used qualitatively to monitor production orpurification of MAP by simply visualizing the released detection moiety.The method can also be used to quantitatively monitor such production orpurification, or measure MAP activity, e.g., in tissue or fluid samples,by quantifying the detection moiety, and comparing the value withcontrols of known MAP activity.

In other embodiments of the invention, methods are provided fordetermining whether a substance inhibits a MAP. These methods areperformed like the method for MAP activity disclosed above, except thata constant amount of MAP is utilized, and the substance is added beforethe MAP is combined with the peptide. Thus, the method comprisescombining the substance, the MAP, and a peptide comprising an N-terminalmethionine under conditions that the N-terminal methionine can becleaved from the peptide by the MAP to produce a cleaved peptide,wherein the peptide contains a C-terminal detection moiety which isreleased by a second peptidase only if the N-terminal methionine hasbeen cleaved from the peptide; reacting any cleaved peptide producedwith the second peptidase to release the detection moiety; and detectingany detection moiety released. If the substance inhibits MAP, there willbe less (or no) detection moiety released from the peptide than if thesubstance does not inhibit MAP. Thus, it is preferred that ano-inhibitor control is utilized in order to be able to determine if thedetection moiety released is less than that released without theinhibitor. It is also preferred that a control be utilized to rule outinhibition of the second peptidase, rather than the MAP. For example,where the second peptidase is dipeptidyl peptidase IV, such a controlwould be the use of the peptide Gly-Pro-p-nitroanilide instead ofMet-Gly-Pro-p-nitroanilide.

This inhibitor-detection method can utilize MAP or the second peptidasefrom any source. However, since some known inhibitors of MAP onlyinhibit some forms of MAP (e.g., fumagillin inhibits type 2, but nottype 1, MAP), the MAP selected for this assay should be similar or thesame as the MAP to which inhibition is desired.

The MAP and second peptidase can be in purified form or in an impureform, such as a cell lysate, provided the other components of the impurepreparation do not interfere with the MAP-peptide or MAP-secondpeptidase reaction. In one variation, the MAP and second peptidase canbe produced in transgenic cells (prepared by methods known in the art)to which the peptide and inhibitor are added.

These methods can be used to qualitatively or quantitatively screen forMAP inhibitors. For example, the methods can be used qualitatively toevaluate whether a substance is a strong MAP inhibitor, where thedifference in released detection moiety between treatments with asought-after inhibitor and treatments or controls without such aninhibitor can be readily ascertained visually. The methods can also beused to quantitatively evaluate the relative inhibitory activity of asubstance, or quantify the amount of an inhibitor in, e.g., tissue orfluid samples, by quantifying the released detection moiety andcomparing the value with controls having known amounts of an inhibitor.For example, the amount of fumigillin in the tissue of a cancer patienttreated with that MAP inhibitor to prevent angiogenesis can be monitoredwith these methods.

These inhibitor-detecting methods can also be utilized to simultaneouslyscreen compounds for inhibitor activity of MAP and the second peptidase,and/or the third enzyme, if so employed. In this scheme, the inhibitoris added with MAP, the second peptidase, and the third enzyme, ifdesired. Any substance inhibiting release of the detection moiety isthen evaluated for inhibition of the second peptidase and/or the thirdenzyme. For example, MAP, dipeptidyl peptidase IV, cathepsin C, and thepeptide Met-Gly-Pro-Gly-Phe-p-nitroanilide can be used to test forinhibitory activity of a substance. If the substance is not inhibitoryto any of these enzymes, p-nitroanilide will be released. However, if areduced amount of p-nitroanilide is released, the substance can then betested using Gly-Pro-p-nitroanilide (a dipeptidyl peptidase IVsubstrate) or Gly-Phe-p-nitroanilide (a cathepsin C substrate). If theinhibitor does not inhibit release of p-nitroanilide in either of thesetests, then the inhibitor is specific for MAP. If the inhibitor does notallow the release of p-nitroanilide when Gly-Prop-nitroanilide was used,but does allow release when Gly-Phe-p-nitroanilide was used, theinhibitor is specific for dipeptidyl peptidase IV; if the result is theopposite, then the inhibitor is specific for cathepsin C.

In additional embodiments of the invention, reaction mixtures areprovided which are useful for either the method for detecting MAP, orthe method for detecting an inhibitor of MAP, the second peptidase, orthe third enzyme. The reaction mixtures comprise a mixture of (a) apeptide comprising an N-terminal methionine which can be cleaved by theMAP, and a C-terminal detection moiety which can be released by a secondpeptidase only if the N-terminal methionine has been cleaved from thepeptide, and (b) the second peptidase. Additionally, the reactionmixtures of this embodiment must be suitable for use as the reactionmixture of the method for detecting MAP described above in that factorssuch as pH and ionic strength must be suitable for activity of the MAPand second peptidase. A preferred second peptidase in this reactionmixture is dipeptidyl peptidase IV. When the second peptidase isdipeptidyl peptidase IV, a preferred peptide comprises Met-X_(aa)-Pro,wherein X_(aa) is Ala, Cys, Gly, or Ser, and the most preferred peptideis Met-Gly-Pro-p-nitroanilide. These reaction mixtures may also comprisea MAP, which is useful for the methods for detecting MAP inhibitors.

In still other embodiments of the invention, kits are provided which areuseful for performing the method for detecting MAP, or the method fordetecting an inhibitor of MAP, the second peptidase, or the thirdenzyme. The kits comprise (a) a peptide comprising an N-terminalmethionine which can be cleaved by the MAP, and a C-terminal detectionmoiety which can be released by a second peptidase only if theN-terminal methionine has been cleaved from the peptide, (b) the secondpeptidase, and (c) instructions for performing the method. A preferredsecond peptidase in these kits are dipeptidyl peptidase IV. When thesecond peptidase is dipeptidyl peptidase IV, a preferred peptidecomprises Met-X_(aa)-Pro, wherein X_(aa) is Ala, Cys, Gly, or Ser, andthe most preferred peptide is Met-Gly-Pro-p-nitroanilide. These kitspreferably also comprise a MAP, which is useful for the methods fordetecting MAP inhibitors, or as a component of a control in methods fordetecting MAP. The components of these kits can be in separatecontainers, or some or all of the components may be mixed together.

In additional embodiments of the invention, peptides are provided whichcomprise an N-terminal methionine which can be cleaved by a methionineaminopeptidase, and a C-terminal detection moiety which can be releasedby a second peptidase only if the N-terminal methionine has been cleavedfrom the peptide. A preferred peptide for these embodiments comprisesMet-X_(aa)-Pro, wherein X_(aa) is Ala, Cys, Gly, or Ser, and the mostpreferred peptide is Met-Gly-Pro-p-nitroanilide.

INDUSTRIAL APPLICATION

The methods and compositions of the present invention are useful fordetecting MAP or MAP inhibitors rapidly and quantitatively, forisolating, purifying, or quantifying new MAPs or inhibitors of MAPs, orfor determining or quantifying MAP activity in a sample.

Preferred embodiments of the invention are described in the followingexample. Other embodiments within the scope of the claims herein will beapparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

EXAMPLE

This example illustrates embodiments of the method for quantifying MAPand the method for detecting an inhibitor of MAP according to theinvention.

To determine whether Met-Gly-Pro-p-nitroanilide is a substrate for MAP,and whether the presence of dipeptidyl peptidase could affect theactivity of MAP, an AccQ-Tag assay was used as described in Zuo et al.,supra. Table 1 shows that this peptide is a substrate for both type 1and type 2 MAP.

TABLE 1 Kinetic parameters for yeast type 1 MAP and human type 2 MAP.Data are reported as mean ± SD. Enzymes k_(cat) (min⁻¹) K_(m) (mM)k_(cat)/K_(m) (mM⁻¹min⁻¹) type 1 MAP 864 ± 25 4 ± 0.7 216 ± 35 type 2MAP 518 ± 35 3 ± 0.5 172 ± 26

The activity of both type 1 MAP and type 2 MAP was not affected by theaddition of dipeptidyl peptidase IV.

MAP activity was next determined by monitoring the release ofp-nitroanilide in a microtiter format. Purified type 1 MAP or type 2 MAP(0.6 μg) and/or 0.001 unit of dipeptidyl peptidase IV was added in 47 μlof buffer H (10 mM Hepes, pH 7.4, 10% glycerol), containing 0.1 M KCland 0.1 mM of Co²⁺ in wells of a 96-well microtiter plate. Afterincubating at 37° C. for 5 min, 2 mM of Met-Gly-Pro-p-nitroanilide wasadded to the mixture to start the reaction. A microtiter plate reader,set at 405 nm to detect released p-nitroanilide, was used to record theprogress of the reactions. As shown in FIG. 2, significant increases inreleased p-nitroanilide occurred only when MAP (either type) is presentalong with dipeptidyl peptidase IV. These curves are highly reproducibleand linear, with correlation coefficients (r²)>0.98.

To demonstrate the use of this method in detecting a type 2 MAPinhibitor, various amounts of fumigillin were added to each reaction.After incubating the inhibitor with MAP solutions at 37° C. for 10 min,2 mM of Met-Gly-Pro-p-nitroanilide was added to start the reaction, andthe release of p-nitroanilide was monitored with a microtiter platereader at 405 nm as described above. The effect of fumigillin ondipeptidyl peptidase IV activity was also tested by usingGly-Pro-p-nitroanilide as the substrate. As shown in FIG. 3, while bothtype 1 MAP and dipeptidyl peptidase IV activity remained unaffected evenin the presence of 50 nM fumagillin, type 2 MAP activity was completelyinhibited by 20 nM fumigillin. These results are very similar to thoseobtained with the AccQ-Tag method (Griffith et al., supra), indicatingthat this is a reliable method for identifying new MAP inhibitors.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantages attained

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

What is claimed is:
 1. A method for detecting methionine aminopeptidase(MAP) activity in a composition comprising: (a) combining thecomposition with a peptide comprising an N-terminal methionine underconditions that the N-terminal methionine can be cleaved from thepeptide by a MAP to produce a cleaved peptide, wherein the peptidecontains a C-terminal detection moiety which is released by a secondpeptidase only if the N-terminal methionine has been cleaved from thepeptide; (b) reacting any cleaved peptide produced in (a) with thesecond peptidase to release the detection moiety; (c) detecting anydetection moiety released (d) correlating any detected moiety releasedwith detecting MAP activity.
 2. The method of claim 1, wherein (a) and(b) are performed in one aqueous reaction mixture.
 3. The method ofclaim 1, wherein the detection moiety is p-nitroanilide.
 4. The methodof claim 1, wherein the second peptidase is dipeptidyl peptidase IV andthe peptide comprises Met-X_(aa)-Pro, wherein X_(aa) is Ala, Cys, Gly,or Ser.
 5. The method of claim 4, wherein the peptide isMet-Gly-Pro-p-nitroanilide.
 6. The method of claim 1, wherein the MAP isa type 2 MAP.
 7. The method of claim 6, wherein the MAP is a human MAP.8. A method for determining whether a substance inhibits a methionineaminopeptidase (MAP) comprising: (a) combining the substance, the MAP,and a peptide comprising an N-terminal methionine under conditions thatthe N-terminal methionine can be cleaved from the peptide by the MAP toproduce a cleaved peptide, wherein the peptide contains a C-terminaldetection moiety which is released by a second peptidase only if theN-terminal methionine has been cleaved from the peptide; (b) reactingany cleaved peptide produced in (a) with the second peptidase to releasethe detection moiety; (c) detecting any detection moiety released (d)correlating any detected moiety released with determining whether asubstance inhibits MAP activity.
 9. The method of claim 8, wherein thedetecting step comprises quantitating the amount of detection moietyreleased.
 10. The method of claim 8, wherein (a) and (b) are bothperformed in one aqueous reaction mixture.
 11. The method of claim 8,wherein the second peptidase is dipeptidyl peptidase IV and the peptidecomprises Met-X_(aa)-Pro, wherein X_(aa) is Ala, Cys, Gly, or Ser. 12.The method of claim 8, wherein the C-terminal detection moiety comprisesp-nitroanilide.
 13. The method of claim 8, wherein the peptide isMet-Gly-Pro-p-nitroanilide.